The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The psychostimulant amphetamine (AMPH), by acting as a DAT substrate, promotes reversal of DA transport (DA efflux) and increases extracellular DA levels, an event of importance for the stimulant properties of AMPH. Our laboratory has previously demonstrated that the SNARE protein syntaxin 1 (STX1) that is enriched in membrane rafts, associates with the DAT N-terminus, and that this association regulates AMPH-induced DA efflux. We have also shown that the membrane raft-associated protein Flotillin-1 (Flot1) is necessary for the localization of DAT to these membrane microdomains. Flot1 knockdown, which displaces DAT from membrane rafts, blunts AMPH-induced DA efflux. This proposal aims to uncover the mechanisms by which DAT posttranslational modifications and DAT protein/lipid interactions regulate DAT function, and importantly, AMPH-induced DA efflux. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key phospholipid that is mainly concentrated in the inner leaflet of the plasma membrane and is enriched in lipid raft domains. In addition to other functions, PIP2 acts as an essential cofactor to mediate protein recruitment and localization. This includes STX1, which is phosphorylated at Ser14 by casein kinase 2 (CK2). We have shown that STX1 binds the DAT N-terminus and that AMPH promotes this interaction. Preliminary results suggest that PIP2 coordinates STX1 phosphorylation, DAT/STX1 associations, as well as DA efflux. These results also suggest that AMPH stimulates CK2-mediated STX1 phosphorylation, a molecular event that we hypothesized to be required for DAT/STX1 interactions as well as DA efflux. Consistent with this hypothesis, our evidence suggest that inhibition of CK2 reduces STX1 phosphorylation and DA efflux. The long-term goals of this research are to understand how AMPH-induced DA efflux is dictated by DAT interactions with STX1 and PIP2, and/or by post-translational modifications of DAT and DAT-associated proteins. These hypotheses will be tested (for the first time) at synaptic release sites. We propose to test our overarching hypothesis through the following specific aims: S.A. #1) To define how PIP2 interacts with the DAT N-terminus and coordinates DAT/STX1 associations. S.A. #2) To determine the role of DAT interacting molecules and their phosphorylation status in AMPH-induced DA efflux.